Moenomycin and process for producing same

ABSTRACT

A high molecular weight amorphous polysaccharide antibiotic composition containing about 1.9 percent by weight phosphorus, 48.5 percent carbon, 7.3 percent hydrogen, 5.1 percent nitrogen and the balance oxygen; having a molecular weight of 68,000 to 70,000, infra-red absorption spectrum maxima, for the sodium salt at 2.95, 3.40, 6.00, 6.53, 7.28, 8.20 and 9.38 Mu and activity in vitro and in vivo against gram positive micro-organisms; and process of preparing same by cultivation of S. bambergiensis, S. ghanaensis, S. ederensis or S. geysiriensis.

O United States Patent [151 3,674,866

Lindner et al. July 4, 1972 m MOENOMYCIN AND PROCESS FOR [58] FieldolSearch ..167/65 AB;195/80;424l118 PRODUCING SAME [5 6] Relerences Cited [72} Inventors: Fritz Lindner; Karl Heinz Wallhlusser,

both of Hofheim/Taunus', Gerhard Huber, OTHER PUBLICATIONS Frankm'mam' Ge'many Miller, The Pfizer Handbood of Microbiac Metabolites, Mc- [73] Assignee: Farbwerke llocchst Aktiengesellschaft vor- Grew-Hill Book Co., Inc., N.Y., N.Y., 196l,p age 594.

mals Meister Lucius 8: Bruning, Frankfurt/Main, Germany Primary Examiner-Jerome D. Goldberg Filed: sept- 1964 Attorney-Curt|s, MOITIS8L Safford [21] Appl. No.: 393,760 ABSTRACT A high molecular weight amorphous polysaccharide antibiotic Rehmd Amman composition containing about 1.9 percent by weight [63] Continuation-impart of Scr. No. 216,751, Aug. 14, phosphorus, 48.5 percent carbon, 7.3 percent hydrogen, 5.1 1962, abandoned, which is a continuation-in-part of percent nitrogen and the balance oxygen; having a molecular Ser. No. 87,750, Feb. 8, 1961, abandoned. weight of 68,000 to 70,000, infra-red absorption spectrum maxima, for the sodium salt at 2.95, 3.40, 6.00, 6.53, 7.28, [30] Foreign Application Priority Data 8.20 and 9.38 and activity in vitro and in vivo against gram positive microorganisms; and process of preparing same by Feb. 10, i960 Germany ..F 30,506 cumvafion of s bambergl-emis, s Shawna, s defends or S 52 use: ..424 118, 195/80 808mm" 8 Claims, No Drawings MOENOMYCIN AND PROCESS FOR PRODUCING SAME The present application is a continuation-in-part of application Ser. No. 2l6,7$l filed Aug. 14, I962, now abandoned, which, in turn, is a continuation-in-part of Application Ser. No. 87,750 filed Feb. 8, I961, now abandoned.

The invention relates to a new antibiotic called Moenomycin, and to a process of preparing it.

This new antibiotic is prepared from the culture of Streptomyces bambergiensis ATCC No. 13,879 which has a certain similarity with Streptomyces glaucus, Streptomyces prarr'nus and Streptomyces hr'rsmus which form green spores, but differs from these as shown in Table l forming part of this application. While Streptomyces glaucus clearly belongs to the spira group (35 spirals) as regards the type of sporophores, one spiral only can be found in Streptomyces bambergienstlr so that this organism is to be ranged in the Rerinaculum apermm group. There are other differences with regard to the pigment formation and color of the vegetative mycelium. Streptomyces prasinus (Ettlinger et al.) probably belongs to the Retinaculum apermm group, too. Owing to its copperred vegatative mycelium on calcium-malate-agar, the lacking liquefaction of gelatin and formation of antibiotics, however, this type is also distinguished from Streptomyces bambergiensis. Like the afore-mentioned organism, Streptomyces hirsmus (Ettlinger et al.) is also not able to produce antibiotics.

It belongs to the spira-group, it does not show casein degradation (peptonization of milk) and decolorizes litmus paper. Since Streptomyces bambergiensis ATCC No. l3,879 is not identical with one of the types described in Bergeys Manual of Determinative Bacteriology, 6th edition (I948) and in the Code of Determination by NY. Krassilnikow, Moscow (i949), it was called Streptomyces bambergiensis owing to its origin from a soil sample taken near Bamberg.

There could be isolated further streptomyces which fonn the same antibiotic viz. Streptomyces ghanaensis ATCC No. l4,672 (from Ghana) which in its properties resembles Streptomyces bambergiensilr, as well as Streptomyces ederensis ATCC No. 15,304 (from the Eder valley) and Streptomyces geysiriensir ATCC No. 15,303 (from an Iceland geyser). The aforementioned streptomyces are compared in Table 2 of this application, said table showing the morphological and physiological properties of these organisms.

The new antibiotic Moenomycin may be obtained by cultivating in an appropriate manner a Streptomyces bambergienxii strain or other equivalent Streptomyces such, for example, as .S'lrepmmyces ghanaensis, Streptomyces ederensis, Streptomyr'es geysiriensr's or the variants or mutants thereof, and isolating the antibiotic from the mycelium and culture solution.

The cultivation takes place under aerobic conditions in an aqueous medium containing, in addition to organic salts, starch, glucose, cane sugar or molasses as source of carbon, as well as soybean groats, comsteep liquor, yeast extract, cotton seed meal, nitrates or ammonium salts as a source of nitrogen, until a maximum antibiotic activity is reached which is proven in the test against Staphylococcus aureus P 209.

The antibiotic is present mainly in the mycelium. Small amounts, however, are present in the culture solution from where they are obtained by extraction with organic solvents such, for example, as butanol, or with the aid of adsorbing agents.

To recover the antibiotic from the mycelium, the latter is separated from the culture solution in an appropriate manner, e. g. by centrifugation, and extracted with an organic solvent. There are preferably used completely miscible or at least partially, i.e., to a degree of about percent miscible solvents such as, for example, alcohols, e.g. methanol, ethanol, butanol; partially etherified alcohols, e.g. ethylene glycol monomethyl ether; ethers, e.g. dioxane; and ketones, e.g. acetone. The extraction can also be carried out by destruction of the cells, for example, by deep-freezing and subsequent thawing, or by homogenizing, with water or aqueous solutions of salts, organic or inorganic acids.

The crude antibiotic can be recovered from the mycelium extracts by evaporating the solvents, preferably under reduced pressure. To separate the resulting residue from inactive impurities, it is extracted by means of a hot polar organic solvent such, for example, as methanol, and the antibiotic is precipitated from the extract and concentrated under reduced pressure with a nonpolar organic solvent such, for example, as diethyl ether, diisopropyl ether or ethyl acetate.

The isolation from the mycelium extract can also be carried out by evaporating the organic solvent, adjusting the pH to a value ranging from 1-5, preferably from 2-3, with the aid of a mineral acid and then extracting the antibiotic from the remaining aqueous residue with a solvent which is not miscible with water, or is only scarcely miscible with water such, for example, as n-butanol. The antibiotic can also be adsorbed in known manner from the aqueous residue by adsorbing agents such, for example, as bleaching earths. The elution is suitably effected by means of mixtures of water and organic solvents such, for example, as mixtures of methanol and water, methyl glycol and water or pyrimidine and water, the water content of which may range between 30 and 70 percent, and is preferably of the order of about 50 percent. The crude antibiotic is obtainable from the extract by evaporation and precipitation as described above.

The crude material thus obtained can be purified in different ways, for instance, by column chromatography on inorganic adsorbents such, for example, as magnesium silicate (FLORISIL magnesium silicate), charcoal or bleaching earths, by gel filtration on cross-linked dextrans showing microfiltering properties, as they are commercially obtainable under the names of SEPHADEX dextrans G 25, G 50 and G 100', or by separation on anion exchange cellulose, such, for example, as ECTEOLA-cellulose and DEAE-cellulose. The material can furthermore be concentrated by salting it out from a concentrated aqueous solution, for instance, by satura tion with ammonium sulfate or by dialysis. In the latter case the antibiotic does not pass through the dialysis membrane. The last-mentioned properties are indicative of the highmolecular weight structure of the antibiotic.

Moenomycin can be further purified by counter-current distribution or by partition-chromatography on suitable adsorbing agents such as kieselguhr, for which procedure the n-butanol/citrate-phosphate-buffer of pH 4 (Mc.llvaire buffer) is, for example, particularly suitable.

The new antibiotic which is distinguished by its properties from the antibiotically effective substances hitherto described in the literature constitutes an amorphous, colorless weak acid that forms the corresponding salt when reacted with one mol of a metal hydroxide or an organic base. Thus, alkali metal salts, alkaline earth metal salts and salts of other metals, e.g. aluminum and zinc, as well as salts of organic bases, particularly nitrogen bases, have been synthesized.

The antibiotic, whether in the form of the acid or in the form of the salts mentioned above, particularly its alkali metal salts, is soluble in water, alcohols of low molecular weight, e. g. methanol, in formamide and dimethyl formamide; it is less soluble in alcohols of two or more carbon atoms, such as ethanol, n-propanol and n-butanol as well as in pyrimidine; it is slightly soluble in ether and ethyl acetate, and it is insoluble in nonpolar organic solvents, such as benzene and chloroform.

The substance is stable in neutral aqueous and methanolic solution. it is slowly decomposed, however, in acid and in alkaline solutions. The sodium salt of Moenomycin in dry solid fonn is stable for at least 48 hours at C.

Upon careful analysis the acid form of the antibiotic was found to contain:

C 48.5 by weight H 7.3 by weight N 5.1 by weight P 1.8 by weight 0 remainder It has no definite melting point, decomposition starting at 200 C and has maximum absorption in the infra-red spectrum at 2.95, 3.41, 5.81, 6.13, 6.45, 7.28, 7.53, 8.22, 9,50 and l0.30 The corresponding values of the maximum absorption ofthe sodium salt are at 2.95, 3.40, 6.00, 6.53, 7.28, 8.10, 9.38 t.

It shows no migration in paper-electrophoresis in buffer solutions of pH L9 and 7.8, but shows an easy anodic migration at pH 9.8. The R; values of the antibiotic in paper chromatography are as follows:

System R,- nbutanol/triethanolamine/methylisobutylketone/water l4:l:l:5 0.05 benzene/glacial acetic acid/water 2:2: I n-butanollglacial acetic acid/water 4i] :5 0.88 t-butanol/glacial acetic acid/water 60:61 0.70 butanol saturated with water 0 sec.butanol/glacial acetic acid/water The following reactions are negative ninhydrine, biuret, Fehling, Ehrlich, Sakaguchi, aniline phthalate, ferric chloride. Moenomycin gives with antimony trichloride and chlorosulfonic acid (as spray reagents) a red-violet color reaction and reacts with potassium permanganate and periodic acid/Schiff reagent.

Potentiometric titration shows an equivalent weight of about 800 and a P -value of 4. l. Determination of the molecular weight by means of an ultracentrifuge and measurement of the light dispersion gives a value of 68,000 to 70,000. Moenomycin is not dialyzable and can be salted out from aqueous solutions with neutral salts, for example, ammonium sulfate.

The maximum absorption in the ultraviolet within the range of from 220-400 mp is at 258 mu, E in water at a pH- value of 7 being 60. The optical activity of Moenomycin is [a] 4'.

Hydrolysis with 2N-hydrochloric acid (3 hours, l00 C) provokes in the paper chromatograph (n-butanol/pyridine/water 413:2 system) at least 5 ninhydrin-positive spots, which, moreover, reduce alkaline silver nitrate solution and must be considered as amino sugars. One of these spots was correlated to glucosamine. Moreover, some neutral sugars are formed. Of these glucose was hitherto identified. With alkaline hydrolysis (0.] N-sodium hydroxide solution, I hour, l00 C) a mixture of phosphoric acid esters (sugar phosphates or aminosugar phosphates) is formed.

With short-time acid hydrolysis (normal hydrochloric acid, minutes, 100" C) a lipoidlike substance separates which can be isolated by extraction with chloroform of ether. Moenomycin is not attacked by the following enzymes: trypsin, papain, pepsin, amylase, hemi-cellulase, lysozyme, phosphorus diesterase (snake venom), alkaline phosphatase, hyaluronidase, n-acetyl-glucosaminidase, riboand desoxyribonuclease.

On the basis of these properties and in view of the fact that it is not dialyzable and can be salted out, Moenomycin can be considered as an acid polysaccharide of high molecular weight containing phosphate ester groups and a lipoid portion. The carbohydrate part consists of neutral and amino-sugars.

Unlike high molecular weight, phosphorus-containing antibiotics heretofore proposed, e.g. hekenin (from Penicillium funicufosum) which is an unstable nucleoproteide, and statolon (from Penicillium slolomferum) which contains arabinose, galactose and galactosarnine as structural units, and both of which are active exclusively on viruses, Moenomycin does not show anti-virus activity, is extraordinarily stable and does not contain protein, nucleic acid, or arabinose, galactose or galactosamine units.

Moenomycin is very well tolerated by wamublooded animals. The LD in mice for oral, subcutaneous or intraperitoneal administration is 2,000 mg/kg; in intravenous administration the LD in mice is [,400 mg/kg. The new antibiotic possesses a high activity in vitro and in vivo against gram-positive micro-organism, especially against staphylococci resistant to tetracycline, penicillin, streptomycin, novobiocin and erythromycin. Table 3 shows the biological activity. When administered subcutaneously three times in amounts of 2 milligrams/kg each, the antibiotic affords a percent protection in the mouse infected with Streptococci haemolytici, as shown in Table 4.

in therapeutic tests on mice infected with tetracycline-resistant staphylococci, it affords a 100 percent protection when administered in a single dose of 4 mg/kg i.p. In the subcutaneous disinfection test according to Fussganger and Rolly (Arzneimittelforschung 6, 260, 1966)), a dose of l-2 mg/20 g of body weight of a mouse causes inhibition of the growth of the tetracycline-resistant staphylococci.

Though Moenomycin has a low activity in vitro against Brucellae, a good activity in vivo was observed in mice infected with brucellosis. Upon intravenous injection of 0.75 trig/20 g of mouse, 90 percent of the mice survived an intraperitoneal infection with Brucellae abortus, whereas of the control animals which were equally infected and were given the same dose of streptomycin, only 60 percent survived. The same effect (90 percent of surviving mice) was attained, when the Moenomycin was administered subcutaneously in a dose of 1 mg/ 20 g of mouse 24 hours prior to the infection.

The following examples illustrate the invention, but they are not intended to limit it thereto, the percentages being by weight.

EXAMPLE I A. Preparation of the inoculation material 80 Milliliters of the nutrient solution described below is introduced into a 300m] Erlenmeyer flask, the solution is adjusted to pH 6.6, sterilized for 30 minutes at 121 C in the autoclave and, after cooling, inoculated with a spore suspension of Srreplomyces Bambergiensis ATCC No. l3,879 obtained by floating off the spores from a culture on a nutrient medium consisting of cats and agar in a Roux flask.

Nutrient solution containing soybean meal I if glucose I if NuCl 0.25 q

The inoculated nutrient solution in the flask is cultivated for 48 hours in a shaking apparatus at 28 C and then used for inoculation of the fermenter both described below.

B. Fermentation The cultivation on a larger scale is effected in a fermenter with a nutrient solution containing Starch sugar 4 Soybean groats 3.4 NaCl 0.25

50 liters of this nutrient solution are sterilized for 30 minutes at l2l C in a IOO-liter fermenter, then cooled to 28 C and seeded with 1-2 percent of the inoculum obtained as described in Part A. The organisms are grown with aeration (50 liters of air per l liter of liquid per hour). During the first 48 hours, the pH value falls from 6.8 to about 6.0 6.2, and then rises slowly to 7.5 8.0 (96 hours).

C. Isolation Upon harvesting, the mycelium is separated from the fermentation solution by centrifugation and extracted while stirred, with ID liters of methanol. The methanol is filtered off and the residue is again extracted with 10 liters of methanol. The combined methanol extracts are evaporated under reduced pressure until the methanol is removed and the aqueous phase obtained (about 500 milliliters) is adjusted twice by means of hydrochloric acid to a pH of 3, and extracted with 500 milliliters of butanol each time. The butanol extract is evaporated under reduced pressure to I00 milliliters. On standing in a refrigerator, crude Moenomycin separates. The

product is filtered ofi' with suction, washed with ethyl-acetate and dried. The yield is 18 grams. The crude product can be obtained from the concentrated butanol solution likewise by precipitation with ether or ethyl-acetate. The product contains about 20 percent of Meonomycin (microbiological test).

D. Purification by dialysis and chromatography on magnesium silicate 20 grams of the crude product prepared as described in Part C are suspended in 200 milliliters of water, the solution is adjusted to a pH of 7 by means of normal sodium hydroxide solution, any undissolved matter is filtered off and the mass is dialyzed for three days against running tap water. The solution remaining in the dialysis vessel is adjusted to a pH of 3 by means of hydrochloric acid extracted twice with 250 milliliters of normal butanol each time. After concentration of the butanol under reduced pressure the enriched Moenomycin is obtained as described in Part C by freezing it out or by precipitation with ether. The yield is 8.5 grams. The brown product, of 20 percent strength, contains 48 percent of Moenomycin.

In order to remove the major part of the brown dyestufi' contained in the crude Moenomycin, the crude product is dissolved in I milliliters of methanol, any undissolved matter is filtered off and the solution is brought on a chromatography column with 80 grams of magnesium trisilicate (FLORISIL) suspended in methanol. The elution takes place with 1 liter of methanol (fraction l) and 500 milliliters of aqueous methanol of 80 percent strength (fraction 2). Upon elimination of the solvent by evaporation, fraction 1 yields 5.4 grams of Moenomycin in the font: of a light amorphous powder, and fraction 2 yields 1 .3 gram of Moenomycin of a slightly reduced activity.

E. Purification by gel-filtration on SEPHADEX l gram of Moenomycin obtained as described in Part D is dissolved in i0 milliliters of water and applied on a chromatography column (80 X 5 cm) containing swollen SEPHADEX. On development with water, l6 fractions of I00 milliliters each are collected and analyzed. According to ultraviolet measurement at 258 my. and determination of the microbiological activity, Moenomycin is present in Fractions 5 to (maximum in Fraction 8), whereas dyestuffs appear in Fraction 4 and in Fractions 1 l to [6. A colorless amorphous product (055 gram) showing a Moenomycin conten of 8085 percent is obtained from Fraction 8 by lyophilization.

F. Purification by chromatography at ion-exchange cellulose 400 milligrams of the Moenomycin purified by gel filtration according to Part E are dissolved in 8 milliliters of water and applied to a chromatography column I00 X 3 cm) with EC- TOELA anion exchange cellulose. The ECTEOLA powder was previously converted into the bicarbonate form by means of an aqueous NH,HCO,,-solution of 5 percent strength and is suspended in the column by means of NH,HCO -solution of4 percent strength. The column is developed by means of water 100 ml) and subsequently by a gradient elution (linear gradient) with 1 liter each of NH,HCO -solution of 0.4 and 10 percent strength, respectively. Fractions of 10 milliliters each are collected and analyzed by ultraviolet measurement (258 my) and microbiological tests. Moenomycin is present in Fractions 8] to l l0. The fractions are combined, the solution is stirred with an excess of AMBERLITE lRC-SO and freed from salt in this manner. By lyophilization of the solution, l80 milligrams of pure, colorless Moenomycin (acid form) are obtained.

G. Purification by means of the zinc salt 5 grams of crude Moenomycin prepared as described in Part C are dissolved in I00 milliliters of water while neutralized to a pH of 7. After separation of the insoluble portion,

I00 milliliters of a ZnSO,-solution of 10 percent strength are added and the solution is adjusted to a pH of 8 by means of normal sodium hydroxide solution. The precipitated zinc salts containing the Moenomycin are removed by centrifugation, washed out with I00 milliliters of water and suspended in I00 milliliters of water. After acidification to a pH of 2 by means of lN-sulfuric acid, the clear solution is extracted twice by means of milliliters of n-butanol each, the butanol extracts are combined, concentrated and the Moenomycin is isolated by precipitation with ethyl-acetate. The yield is 1.9 grams, Moenomycin content 45 percent.

H. Potassium salt of Meonomycin 0.5 gram of pure Moenomycin (acid form, obtained as described in Part F) is dissolved in 5 milliliter of water and the solution is adjusted to a pH by means of 0.5-potassium hydroxide solution. By lyphilization 0.52 gram of the potassium salt of Moenomycin is obtained.

1. Sodium salt of Moenomycin 300 milligrams of pure Moenomycin (acid form. obtained as described in Part F) are dissolved in l milliliter of methanol, the solution is adjusted to a pH of 7 by means of methanolic sodium hydroxide solution and the sodium salt is precipitated by addition of ether, washed with water and dried under reduced pressure. The yield is 240 milligrams.

K. Diethylamine salt of Moenomycin 250 milligrams of pure Moenomycin (acid form, obtained according to Part F) are dissolved in 3 milliliters of water and 2 milliliters of an aqueous diethylamine solution of 50 percent strength are added. The diethylamine salt of the Moenomycin formed is isolated by evaporation of the solution under reduced pressure.

L. Cyclohexylamine salt of Moenomycin To a solution of 450 milligrams of pure Moenomycin in 10 milliliters of methanol 300 milligrams of cyclohexylamine are added and the salt that has formed is isolated by evaporation of the solution under reduced pressure.

EXAMPLE 2 A. Preparation of the inoculum 100 cc of a nutrient solution consisting of l percent glucose, l percent of casein peptone, 0.2 percent of beef broth and 0.25 percent of NaCl, pH 7.0, in an Erlenmeyer flask of 300 cc capacity, are innoculated with spores of a soil culture of the strain Srrepromyces ghanaensis, ATCC No. l4,672, and shaken for 48 hours at 28 C. This culture serves as inoculum for the fermentation.

B. Fermentation Fermentation is effected with 10 l of a nutrient solution having the following composition:

3 percent starch sugar (glucose) 1 percent coarse grain soybean flour 1 percent cornsteep liquor 0.2 percent oat flakes 0.5 percent CaCO 0.2 percent KNO 0.02 percent zinc sulfate pH 7.5. After having sterilized this nutrient solution, 300 cc of the inoculum prepared as described in Part A are added to the above solution and fermentation is allowed to proceed at 3245 C, while stirring and aerating well. During the fermentation, 100-250 cc of sperm oil are added as an antifoaming agent. When the optimum activity is reached (after 72-l20 hours) the product is collected from the batch and isolated as described in Example I.

TABLE I [Comparison of Stroptomycos forming green spores as described in the literature] S. bamberg.

A'ICC. N n S. virido- S. pmsino' Characteristics 18 87'.) chro m b. glauc-us S. z'irida'ns h. albozziridia S. prasinm" S. hirsutm" piloais' Sporophnre. Rutinaculum Spira (bis 10).. Spiro (3 5).. Spirit (5 12). Spira (34) Spire 11.1 Spira (3).... Spira (k3).

"-P- upcrtl i. Hlmpuulspnrt-s ()v11l,l\airy Oval, tlm|'ny. ()v:tl tlylindricalnn tyliudrical. Oblong-oval 0val,tllorny Oval, hairy.

thorny.

TABLE 1 (Comparison oi Streptomyces forming green spores as described in the literature] S. bambcrp. ATCC. No S. vmdov v I ,5, m Characteristics 13 B79 chro'm' S. glaucus' .8. rmdam' S, ulbot-zrldn pmsinua" S. hirautua" pilnsia' Color oi spores Green-dark Light green, Green-grey. Dark grey- Greenish-grcy... Green-dark Dark green. Dark Gr green. grey green, grey green. greenv smoke blue. Vegetative rnycelium... Cream-light Green brown.. Colorlcss. Green brown. Green-brown... Light brown Colorless- Brick-red.

brown. white Sol ble plgment Brown. Brown... Brown. reen Melanlne pigment... Starch hydrolysis Caseinh drolysis.... +(weak).. Gelatin dation..... +(slow) +(slow). m Nitrater uction.... Litmus Blue Blue R Ca-malate-agar (color of Cream Copper-rid... d,

the vegetat. mycellnm) Antibiotic eiiicacy Present..... Lacking or Present..... Nont- Zwnc.......... .\one.,,. w ak Weakagainst grampositive Bacteriae.

According to N. A. Krassilnlkow. "According to Ettlinger et Bl.

TABLE 2 (Morphological and physiological properties oi new Strcptomycesl Culture medium Properties .5. ederemia ATCC No. i5 304 bambemienaia A'ICC No. 13 879 S. oeyairlemia ATCC No. 16 303 S. gllanumla A'ICC No.14 672 Myccliurn Aerial myce-liurn and spores. Soluble pigment.

Bouillon-gelatin Liquefaction Growth veg Mycelium. Starch-agar Sol, pigment Starch degm lat.

Growth veg.

. Aerial m ccliurn and spores...

Dark brown... Weakly white. Dark brown Growth veg lilucosengar yer-Hum Aerial mycellum and spores... ircyqvhite. White Sol. pigment. Dark brown. Growth veg... .\Iycellum Light brown. Li ht brown. synthetic ugar. Aerial mycelium and spores... None. Pa 0 brown.

Sol. pigment Light brown. Light brown. Nitrate reduct Growth veg...

Zilycelium. Cantabile-agar Sol. plgmenL. Dark brown Malatc degrad (under the colony)..

Growthveg.... Glucose asparilc agar. Mycelium Aerial mycelilun anti nbore.

Aerial rnycellurn and sporcs..

Dark brown.

Hreyhrown.

Sol. pigment Growth veg... l Nutrient BRBL \lycellum Dark brown...

Aerial mycellum and sp0res None Sol. pi i: Dark brown...

Growt Emerson agan. Mycelinm Dark brown Aeflal mycelium and spores. None Sol. pi ent Dark brown Growl; veg..

Cellulose agar.. Mycellum Grey-brown.

Aerial rnycellum and spores. .l None l Sol. pigment Dark brown...

Cellulose degradat Growthveg Mycellum brown. Caseln-litmusagar Aerial mycelium and spores Litmus-colour Casein degradation- Peptone-agar slant.

ol. pigment Light grey lla'iiii'iai lalc yellow.

Yellow-cream.

Light grey (ream Whiir'.

. Urecn.

. Light brown Whlic.

Yellow cream. Brownlsh white.

++ Yeilowrream. G reen.

. Cream.

White green.

C ream. White.

. Cream.

Green.

.. Cream.

White-light grey. Blu

. Green.

Sporophore-type.

Spores....

. Rectus........

Retlnaculum apertnrn (Rectus).

. Oval, smooth..

Oval, hairy.

Splra (Monovert- S l p m). Cylindrical. hairy...

Retlnaculum apcrtum. Oval, thorny.

N01L-K=diameter oi colony in m m.; Z=dlarneter of zone in mm.

TABLE 3 Antibacterial spectrum of Moenomycin TABLE 4 Therapeutic activity of Moenomycin in mice infected with SITPPIUIOCCUX lmemolytirus Infection: intraperitoneally Treatment: 3 doses of Moenomycin immediately, 4 and 20 hours afler the infection.

Total dose Number of Surviving animals after ,g/kg animals 2 days days 3 4 4 3 0 (control) 8 We claim:

1. An antibiotic characterized by the following properties:

a. Elemental analysis C 48.5 by weight H 7.3 by weight N 5.1% by weight P 1.9 by weight 0 remainder.

b. Molecular weight: 68,000 to 70,000.

c. Infra red spectrum of the sodium salt: Maxlma at 2.95,

3.40, 6.00, 6.53, 7.28, 8.20 and 9.38 p.

d. Melting point: No definite melting point, decomposition starting at 200 C.

e. Paper-electrophoresis: No migration in buffer solutions of pH 1.9 and 7.8, anodic migration in buffer solution of pH 9.8.

f. Solubility: Soluble in water, low molecular weight alcohols and polar solvents.

g. Ultra-violet spectrum: Maximum at 258 a, E 60 (water, pH 7) or physiologically compatible salts thereof. 2. A pharmaceutical composition which comprises an antibacterially effective amount of the antibiotic as defined in claim 1 and a phan'naceutical carrier.

3. A process for producing the antibiotic as defined in claim 1 which comprises cultivating a Streptomyces strain of the group consisting of bambergi'ensr's (ATCC No. 13,879), glianaensis (ATCC No. 14,672), ederensis (ATCC No. 15,304) and geysiriensis (ATCC' No. 15,303) under submerged and aerobic conditions in an aqueous nutrient medium until substantial antibacterial activity is imparted to said medium and thereupon extracting an antibiotic from the resulting mycelium and culture medium.

4. Process of claim 3 wherein the antibiotic is isolated from the mycelium by extraction with an at least partially watermiscible organic solvent.

5. Process of claim 3 wherein the antibiotic-forming strain is Streptomyces bambergiensr's (ATCC No. 13,879).

6. Process of claim 3 wherein the antibiotic-forming strain is Strepromyces glmnaensis (ATCC No. 14.672

7. Process of claim 3 wherein the antibiotic-forming strain is Streptomyces ederensi's (ATCC No. 15,304).

8. Process of claim 3 wherein the antibiotic-forming strain is Srrepromyces geysin'ensis (ATCC No. 15,303). 

2. A pharmaceutical composition which comprises an antibacterially effective amount of the antibiotic as defined in claim 1 and a pharmaceutical carrier.
 3. A process for producing the antibiotic as defined in claim 1 which comprises cultivating a Streptomyces strain of the group consisting of bambergiensis (ATCC No. 13,879), ghanaensis (ATCC No. 14,672), ederensis (ATCC No. 15,304) and geysiriensis (ATCC No. 15,303) under submerged and aerobic conditions in an aqueous nutrient medium until substantial antibacterial activity is imparted to said medium and thereupon extracting an antibiotic from the resulting mycelium and culture medium.
 4. Process of claim 3 wherein the antibiotic is isolated from the mycelium by extraction with an at least partially water-miscible organic solvent.
 5. Process of claim 3 wherein the antibiotic-forming strain is Streptomyces bambergiensis (ATCC No. 13,879).
 6. Process of claim 3 wherein the antibiotic-forming strain is Streptomyces ghanaensis (ATCC No. 14,672).
 7. Process of claim 3 wherein the antibiotic-forming strain is Streptomyces ederensis (ATCC No. 15,304).
 8. Process of claim 3 wherein the antibiotic-forming strain is Streptomyces geysiriensis (ATCC No. 15,303). 